Compositions of adult disc stem cells for the treatment of degenerative disc disease

ABSTRACT

This invention provides an isolated disc stem cell population, compositions, and methods of obtaining and growing the same. Moreover, this invention provides an isolated discosphere, compositions, and methods of obtaining and growing the same. An artificial disc containing the cells of the present invention is provided together with methods of making the same. This invention also provides a method of treating a subject having a herniated disc utilizing the cells and methods of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application Ser.No. 60/929,792, filed Jul. 12, 2007, which is hereby incorporated byreference in its entirety.

FIELD OF INVENTION

This invention provides: nucleus pulposus stem cells and methods ofobtaining and growing the same.

BACKGROUND OF THE INVENTION

Back pain resulting from degenerative disc disease is a major cause ofmorbidity, disability, and lost productivity. Back pain is the mostfrequent cause of activity limitation in people under the age of 45, thesecond most frequent reason for physician visits, the fifth-rankingreason for hospitalization, and the third most common reason forsurgical procedures. Additionally, chronic back conditions that are bothcommon and debilitating occur in 15 to 45 percent of people each year,and in 70 to 85 percent of people at some time in their lives. Thefinancial impact in terms of health care dollars and lost work hours tosociety is between $20 billion and $50 billion per year in the UnitedStates alone.

Despite the continued improvements in non-operative and operativetreatment options for patients with lower back pain secondary todegenerative disc disease, no treatment modalities have served as the“magic bullet” to eliminate or consistently improve this condition.Today, however, there are new and exciting opportunities for thedevelopment of treatment modalities derived from the merging ofbiomedical engineering and molecular science. We are closer today thenever before to creating new treatment modalities and devices for thetreatment of degenerative disc disease. Recent examples of advancementsin bioscience and the effect on clinical spine disease include thedevelopment of fusion proteins, total disc arthroplasty and morerecently nucleus arthroplasty. Fusion proteins, such as recombinanthuman bone morphogenetic protein-2 (rhBMP-2), are genetically producedproteins that have the ability to stimulate new bone growth to allow fora more reliable and rapid fusion of spinal vertebrae in the context ofsurgical reconstruction.

The first total disc arthroplasty was performed by Fernstorm in the late1950's. Although initially there was a short period of symptom relief,the prosthesis ultimately failed secondary to subsidence of the implantwithin the spine verebra. Although total disc arthroplasty for thelumbar spine has been performed in Europe since the late 1980's, its usein the United States did not begin until March of 2000 with theintroduction of the S B Charité III (DePuy Spine, Raynham, Mass.). 10,11Several other lumbar spine prostheses have since been introduced,including the Maverick (Medtronic Sofamor Danek, Memphis, Tenn.), thePropisc-L (Spine Solutions/Synthes, Paoli, Pa.), and FlexiCore (StrykerSpine, Allendale, N.J.). Each of these prostheses differs in design withrespect to bearing surface, fixation to bone, number of articulations,material, constraint, and mobility of the center of rotation. Inaddition to the lumbar disc arthroplasty, as of last year trials forcervical disc arthroplasty have begun in the United States. Models ofcervical disc arthroplasty include the Bryan Cervical Disk (MedtronicSofamor Danek), the Prestige ST (Medtronic Sofamor Danek), the PorousCoated Motion artificial cervical disk (Cervitech, Rockaway, N.J.), andthe Propisc-C (Spine Solutions/Synthes).

Nucleus arthroplasty or nucleus replacement devices for degenerativespine disease such as the PDN® Prosthetic Disc Nucleus are similar inconcept to TDA and have shown successful results. The PDN® deviceconsists of a hydrogel core center encased in a polyethylene sleevewhich shrinks and swells during normal loading and unloading allowingfor restoration of disc space height and thus mimicking healthy humandisc.

Although the total disc arthroplasty and nucleoplasty may serve as analternative to interbody spinal fusion, the procedure is not without itscomplications. The most common complications include adjacent levelspinal disease, subsidence, and facet joint arthrosis. Furthermore,recent studies from clinical trials have demonstrated incidences ofinfection, vertebral body fracture, implant malposition, subsidence,mechanical failure, and paravertebral heterotopic ossification. Moreserious complications, including anterior dislocation of the implant,have been reported. Also, the issue of wear particles from the totaldisc arthroplasty (TDA) and the potential effects on the spinal cord arestill not known. It is therefore evident that although the developmentof the total disc arthroplasty is a step forward in the treatment ofdegenerative disc disease, the ultimate goal should be the developmentand replacement of a degenerative disc with a new biologic disc whichdoes not have the complications associated with mechanical parts.

More than one million spine surgery procedures are performed annually inUnited States. Furthermore, the lumbar fusion segment of the spinesurgery market is estimated at well over $1 billion in annual revenue.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, an isolated disc stem cellpopulation.

In another embodiment, the present invention provides a method ofisolating disc stem cells, comprising the steps of plating a disc stemcell in a serum free media and producing a discosphere comprisingnucleus pulposus cells, thereby isolating disc stem cells.

In another embodiment, the present invention provides a compositioncomprising disc stem cells

In another embodiment, the present invention provides an isolateddiscosphere.

In another embodiment, the present invention provides a compositioncomprising a discosphere.

In another embodiment, the present invention provides an artificial disccomprising nucleus pulposus cells.

In another embodiment, the present invention provides a method ofproducing an artificial disc, comprising the step of growingdiscospheres in a disc scaffold, thereby producing a spinal discreplacement device.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc, comprising the step ofadministering to said subject an artificial disc comprising nucleuspulposus cells, thereby treating a subject having a herniated disc.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1 shows a microscopic histomorphological assessment with varioustissue stains of 3 month intervertebral disc cultures after in vitrotransplantation of human disc stem cells into evacuated rabbit nucleuspulposus with bony end plates. Panel 1 shows photomicrographs ofhematoxylin-eosin staining of rabbit disc tissue in micrographs A, C,and E (control) and cultured intervertebral disc in micrographs B, D,and F. Magnification: 1.25× (A and B), 10× (C and D), and 20× (E and F).Photomicrographs C and D show the transition zone between the innernucleus pulposus and outer annulus. Photomicrograph E and F show theinner zone of the nucleus pulposus and individual nucleus pulposuscells. Panel 2 shows photomicrographs of safranin staining of rabbitdisc tissue in micrographs G, I, and K (control) and culturedintervertebral disc in micrographs H, J, and L. Magnification: 1.25× (Gand H), 10× (I and J), and 20× (K and L). Photomicrograph I and Jdemonstrate the transition zone between the inner nucleus pulposus andouter annulus. Photomicrograph K and L demonstrate the inner zone of thenucleus pulposus and individual nucleus pulposus cells. Panel 3 showsphotomicrographs of Von Kossa staining of rabbit disc tissue inmicrographs M, O, and Q (control) and cultured intervertebral disc inmicrographs H, J, and L. Magnification: 1.25× (M and N), 10× (O and P),and 20× (Q and R). Micrographs O and P demonstrate the transition zonebetween the inner nucleus pulposus and outer annulus. Micrographs Q andR demonstrate the inner zone of the nucleus pulposus and individualnucleus pulposus cells.

FIG. 2 shows a microscopic histomorphological assessment of theexpression of collagen type II in 3-month intervertebral disc culturesafter in vitro transplantation of human disc stem cells into evacuatedrabbit nucleus pulposus with bony end plates. Photomicrographs ofimmunostaining for collagen type 2 of (A, C, E) control (rabbit disctissue) and (B, D, F) cultured intervertebral disc. Magnification: 1.25×(A and B), 10× (C and D), and 20× (E and F). Micrographs C and Ddemonstrate the transition zone between the inner nucleus pulposus andouter annulus. Micrographs E and F demonstrate the inner zone of thenucleus pulposus and individual nucleus pulposus cells.

FIG. 3 shows a microscopic histomorphological assessment of theexpression of collagen type I in 3 month intervertebral disc culturesafter in vitro transplantation of human disc stem cells into evacuatedrabbit nucleus pulposus with bony end plates. Photomicrographs ofimmunostaining for collagen type I of (A, C, E) control (rabbit disctissue) and (B, D, F) cultured intervertebral disc (annulus matrixprepared in which the nucleus pulposus has been chemically removed andhuman disc stem cell preparations have been introduced). Magnification:10.25× (A and B), 10× (C and D), and 20× (E and F). Micrographs C and Ddemonstrate the transition zone between the inner nucleus pulposus andouter annulus. Micrographs E and F demonstrate the inner zone of thenucleus pulposus and individual nucleus pulposus cells.

FIG. 4 shows a microscopic histomorphological assessment of theexpression of Ki-67 in 3 month intervertebral disc cultures after invitro transplantation of human disc stem cells into evacuated rabbitnucleus pulposus with bony end plates. Photomicrographs ofimmunostaining for Ki-67 of (A, C, E) control (rabbit disc tissue) and(B, D, F) cultured intervertebral disc (annulus matrix prepared in whichthe nucleus pulposus has been chemically removed and human disc stemcell preparations have been introduced). Magnification: 1.25× (A and B),10× (C and D), and 20× (E and F). Micrographs C and D demonstrate thetransition zone between the inner nucleus pulposus and outer annulus.Micrographs E and F demonstrate the inner zone of the nucleus pulposusand individual nucleus pulposus cells.

FIG. 5 depicts a schematic of the intervertebral disc culture systemwith bony end plates. A-Single cell cultures are prepared in media andconditions that promote growth of discospheres (disc stem cellclusters). Discospheres are then prepared and injected into the annulusof a healthy rabbit in which all cells and nucleus pulposus tissue areremoved from the disc. B-Intervertebral disc annulus with bony endplates are then put into a culture vessel with media and growth factors.At the end of 3 months, disc stem cells fill the previously emptyannulus with a disc like structure.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides an isolated disc stemcell population. In another embodiment, the present invention provides adisc stem cells enriched population of cells that can form discospheres.In another embodiment, the present invention provides a disc stem cellsenriched population of cells that can give rise to disc progenitorcells. In another embodiment, the isolated disc stem cell population ofthe present invention comprises a human disc stem cell population. Inanother embodiment, the isolated disc stem cell population of thepresent invention comprises a non-human disc stem cell population. Inanother embodiment, the isolated disc stem cell population of thepresent invention comprises a mammal disc stem cell population. Inanother embodiment, an isolated disc stem cell of the present inventionis derived from a nucleus pulposus of a subject. In another embodiment,nucleus pulposus cells comprise disc stem cells.

In another embodiment, the stem cells enriched cell population of thepresent invention comprises a human disc stem cell population. Inanother embodiment, the stem cells enriched cell population of thepresent invention comprises a non-human disc stem cell population. Inanother embodiment, the stem cells enriched cell population of thepresent invention comprises a mammal disc stem cell population. Inanother embodiment, the stem cells enriched cell population of thepresent invention is derived from a nucleus pulposus of a subject. Inanother embodiment, nucleus pulposus cells comprise disc stem cells.

In another embodiment, a nucleus pulposus is a jelly-like substance inthe middle of the spinal disc. In another embodiment, the nucleuspulposus comprises chondrocytes, collagen fibrils, and proteoglycanaggrecans that have hyaluronic long chains which attract water.

In another embodiment, nucleus pulposus cells of the present inventioncomprise autograft nucleus pulposus cells. In another embodiment,nucleus pulposus cells of the present invention comprise allograftnucleus pulposus cells. In another embodiment, nucleus pulposus cells ofthe present invention comprise xenograft nucleus pulposus cells.

In another embodiment, nucleus pulposus cells of the present inventioncomprise disc stem cells. In another embodiment, nucleus pulposus cellsof the present invention comprise disc progenitor cells. In anotherembodiment, nucleus pulposus cells of the present invention comprisemature disc cells. In another embodiment, nucleus pulposus cells of thepresent invention comprise terminally differentiated disc cells.

In another embodiment, the present invention provides a method ofisolating disc stem cells, comprising the step of producing adiscosphere culture. In another embodiment, the present inventionprovides a method of isolating disc stem cells, comprising the step ofplating nucleus pulposus cells in a serum free media. In anotherembodiment, the present invention provides a method of producing asphere comprising nucleus pulposus cells, comprising the step of growinga culture of nucleus pulposus cells in a serum free media, therebyproducing a discosphere. In another embodiment, the present inventionprovides that a discosphere comprising nucleus pulposus cells is afree-floating structure generated by nucleus pulposus stem cells invitro. In another embodiment, the present invention provides that adiscosphere is a free-floating structure generated by nucleus pulposusprogenitor cells in vitro. In another embodiment, the present inventionprovides that a discosphere is a free-floating structure generated bynucleus pulposus stem and progenitor cells in vitro.

In another embodiment, a disc stem cell of the present invention isdefined by its ability or capacity to form a discosphere. In anotherembodiment, these disc stem cells when grown in adherent culture havethe capability to differentiate, under appropriate differentiatingconditions, to mature or fully differentiate. In another embodiment,fully differentiated nucleus pulposus cells secrete extra cellularmatrix components. In another embodiment, the terms “differentiate” or“differentiation” intended to refer to the development of cells withspecialized structure and function from unspecialized or lessspecialized precursor cells, and includes the development of cells thatpossess the structure and function of nucleus pulposus cells fromprecursor cells. In another embodiment, the terms “differentiate” or“differentiation” intended to refer to the development of cells withspecialized structure and function from disc stem cells. In anotherembodiment, the terms “differentiate” or “differentiation” intended torefer to the development of cells with specialized structure andfunction from disc progenitor cells. In another embodiment, appropriatedifferentiating conditions comprise a media comprising serum.

In another embodiment, the methods of the present invention provide thatdisc material is obtained from the nucleus pulposus of a subject. Inanother embodiment, the methods of the present invention provide thatdisc material is obtained surgically and processed in the lab to createa single cell suspension of nucleus pulposus cells (Example 1). Inanother embodiment, the methods of the present invention provide thathuman disc material is obtained surgically and processed in the lab tocreate a single cell suspension of nucleus pulposus cells. In anotherembodiment, the methods of the present invention provide that humannucleus pulposus is obtained surgically and processed in the lab tocreate a single cell suspension of nucleus pulposus cells.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained by scraping a nucleus pulposus of a subject. Inanother embodiment, heterogeneous population of nucleus pulposus cellscomprises disc stem cells, disc progenitor cells, and differentiatednucleus pulposus cells. In another embodiment, a heterogeneouspopulation of nucleus pulposus cells is scraped from a nucleus pulposusof a human subject. In another embodiment, the present inventionprovides that plating a heterogeneous population of nucleus pulposuscells in a serum free media at low cell density results in the survivalof nucleus pulposus stem cells. In another embodiment, the term survivalof nucleus pulposus stem cells refers to nucleus pulposus stem cellsability to maintain viability under conditions which include aserum-free cell culture media. In another embodiment, the presentinvention provides that the nucleus pulposus cells (majority of thecells in the tissue) die away because they cannot tolerate serum-freeconditions, but the disc stem cells (or nucleus pulposus stem cells,minority of the cells in the tissue) grow into discospheres under thesesconditions.

In another embodiment, the present invention provides that plating aheterogeneous population of nucleus pulposus cells in a serum free mediaat low cell density results in isolation of nucleus pulposus stem cells.In another embodiment, the present invention provides that plating aheterogeneous population of nucleus pulposus cells in a serum free mediaat low cell density results in enriching a nucleus pulposus cellpopulation for disc stem cells. In another embodiment, the presentinvention provides that plating a heterogeneous population of nucleuspulposus cells at low cell density in a serum free media, comprising asubstance the interferes with cell attachment results in the survival ofnucleus pulposus stem cells. In another embodiment, the presentinvention provides that plating a heterogeneous population of nucleuspulposus cells at low cell density in a serum free media, comprisingmethylcellulose which interferes with cell attachment, results in thesurvival of nucleus pulposus stem cells.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus minced inpieces. In another embodiment, the pieces are 0.5-10 mm in size. Inanother embodiment, the pieces are 0.5-20 mm in size. In anotherembodiment, the pieces are 0.5-3 mm in size. In another embodiment, thepieces are 3-6 mm in size. In another embodiment, the pieces are 6-12 mmin size. In another embodiment, the pieces are 12-20 mm in size. Inanother embodiment, the pieces are 1-6 mm in size. In anotherembodiment, the pieces are 3-5 mm in size. In another embodiment, thepieces are 3-4 mm in size (Example 1).

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus by treatingnucleus pulposus with a collagenase II solution (Example 1). In anotherembodiment, a heterogeneous population of nucleus pulposus cells isobtained from a biopsy specimen of nucleus pulposus by treating nucleuspulposus with a 0.1%-1% clostridial collagenase (Worthington CLS II, 140u/mg). In another embodiment, a heterogeneous population of nucleuspulposus cells is obtained from a biopsy specimen of nucleus pulposus bytreating nucleus pulposus with a collagenase II solution followed byplacing the specimen in a shaker thus obtaining a heterogeneouspopulation of nucleus pulposus cells.

In another embodiment, a heterogeneous population of nucleus pulposuscells is obtained from a biopsy specimen of nucleus pulposus byaspiration of a disc of a patient. In another embodiment, aheterogeneous population of nucleus pulposus cells is obtained from abiopsy specimen of nucleus pulposus by aspiration of a disc of a donoranimal. In another embodiment, a heterogeneous population of nucleuspulposus cells is obtained from a biopsy specimen of nucleus pulposus byaspiration of a nucleus pulposus of a donor mammal. In anotherembodiment, a heterogeneous population of nucleus pulposus cells isobtained from a biopsy specimen of nucleus pulposus by aspiration of ahealthy disc of a patient.

In another embodiment, the present invention provides a method ofproducing a discosphere, comprising the step of growing a culture ofnucleus pulposus cells in a serum free media, thereby producing adiscosphere. In another embodiment, the present invention provides thatgrowing a primary culture of nucleus pulposus cells in a serum freemedia results in selecting nucleus pulposus stem cells. In anotherembodiment, the surviving isolated culture of nucleus pulposus stemcells gives rise to discospheres of the present invention. In anotherembodiment, the surviving disc stem cells enriched culture of nucleuspulposus stem cells gives rise to discospheres of the present invention.

In another embodiment, the supplemented serum free media of the presentinvention enables only nucleus pulposus stem cells to grow. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population is produced when grown ina growth factor supplemented serum free media of the present invention.In another embodiment, the methods of the present invention provide thatan enriched nucleus pulposus stem cell population of the presentinvention comprises at least 60% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 70% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 80% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 85% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 90% nucleus pulposus stem cells. In anotherembodiment, the methods of the present invention provide that anenriched nucleus pulposus stem cell population of the present inventioncomprises at least 95% nucleus pulposus stem cells.

In another embodiment, a discosphere is derived from a single nucleuspulposus stem cell. In another embodiment, only disc stem cells growwhen nucleus pulposus cells are plated in a serum free media. In anotherembodiment, only disc stem cells grow when nucleus pulposus cells areplated at low cell density. In another embodiment, only disc stem cellsgrow when nucleus pulposus cells are plated at low cell density in aserum free media. In another embodiment, only nucleus pulposus stemcells can grow as free floating solitary cells in the absence of serum.

In another embodiment, the present invention further provides that discstem cells are grown in a serum free media comprising a compound whichinhibits cell maturation. In another embodiment, the present inventionfurther provides that disc stem cells are grown in a serum free mediacomprising FGF which inhibits cell maturation. In another embodiment,the present invention further provides that disc stem cells are grown ina serum free media comprising a compound that maintains cell juvenility.

In another embodiment, the present invention further provides that discstem cells are grown in a media comprising a TGF-β superfamily member.In another embodiment, the present invention further provides that discstem cells are grown in a media comprising a BMP. In another embodiment,the present invention provides that a BMP of the invention inhibitsdifferentiation (Id) genes.

In another embodiment, the present invention further provides that discstem cells are grown in a media comprising an IL6 cytokine familymember. In another embodiment, the present invention further providesthat disc stem cells are grown in a media comprising leukemia inhibitoryfactor (LIF).

In another embodiment, the present invention further provides that discstem cells are grown in a serum free media comprising a compound whichpromotes cell proliferation. In another embodiment, the presentinvention further provides that disc stem cells are grown in a serumfree media comprising EGF which promotes cell proliferation. In anotherembodiment, the present invention further provides that disc stem cellsare grown in a serum free media comprising interleukin-2 (IL-2). Inanother embodiment, the present invention further provides that discstem cells are grown in a serum free media comprising interleukin-6(IL-6). In another embodiment, the present invention further providesthat disc stem cells are grown in a serum free media comprising a stemcell factor (SCF). In another embodiment, the present invention furtherprovides that disc stem cells are grown in a serum free media comprisingleukemia inhibitory factor (LIF). In another embodiment, the presentinvention further provides that disc stem cells are grown in a serumfree media comprising transforming growth factor-β (TGF-β). In anotherembodiment, the present invention further provides that disc stem cellsare grown in a serum free media comprising a compound that inhibits celldifferentiation (Example 1 and materials and methods).

In another embodiment, disc stem cells of the present inventionproliferate and give rise to additional stem cells. In anotherembodiment, disc stem cells of the present invention proliferate andgive rise to disc progenitor cells. In another embodiment, disc stemcells of the present invention proliferate thus forming a discosphere.In another embodiment, a discosphere of the present invention comprisesnucleus pulposus stem cells and nucleus pulposus progenitor cellsarranged in a circular-spherical structure. In another embodiment, adiscosphere is a ball of cells in which a single disc stem cell givesrise to clones of itself (symmetric division) and to progenitor cells.In another embodiment, a discosphere of the present invention comprisesfree floating nucleus pulposus stem cells and nucleus pulposusprogenitor cells arranged in a circular-spherical structure. In anotherembodiment, the nucleus pulposus cells comprising a discosphere areattached to each other.

In another embodiment, the terms “nucleus pulposus stem cells” and “discstem cells” are used interchangeably. In another embodiment, the terms“nucleus pulposus progenitor cells” and “disc progenitor cells” are usedinterchangeably.

In another embodiment, the term “discosphere” comprises a ball of cellsin which a single disc stem cell gives rise to clones of itself(symmetric division) and to progenitor cells. In another embodiment, theterm “progenitor cells” refer to immature stem-like cells with plasticpotential and high proliferation rates, which can give rise to most ifnot all terminally differentiated tissue cells, but is not by definitiona disc stem cell.

In another embodiment, the methods of the present invention provide thata single cell suspension is prepared for isolating a disc stem cell bycreating certain environmental conditions. In another embodiment, themethods of the present invention provide that a single cell suspensionis prepared for producing a discosphere by creating certainenvironmental conditions.

In another embodiment, the methods of the present invention provide thata single cell suspension is incubated in a humidified Incubator at 37°C. In another embodiment, the methods of the present invention providethat a single cell suspension is incubated in a humidified Incubator at35° C. In another embodiment, the methods of the present inventionprovide that a single cell suspension is incubated in a humidifiedIncubator at 36° C. In another embodiment, the methods of the presentinvention provide that a single cell suspension is incubated in ahumidified Incubator at 38° C. In another embodiment, the methods of thepresent invention provide that a single cell suspension is incubated ina humidified Incubator at 39° C. In another embodiment, the methods ofthe present invention provide that a single cell suspension is incubatedin a humidified Incubator at 40° C. In another embodiment, the methodsof the present invention provide that a single cell suspension isincubated in a humidified Incubator at 41° C. In another embodiment, themethods of the present invention provide that a single cell suspensionis incubated in a humidified Incubator at 42° C.

In another embodiment, the methods of the present invention provide thata single cell suspension is incubated in an incubator furthermaintaining 3-8% CO₂. In another embodiment, the methods of the presentinvention provide that a single cell suspension is incubated in anincubator further maintaining 4% CO₂. In another embodiment, the methodsof the present invention provide that a single cell suspension isincubated in an incubator further maintaining 5% CO₂. In anotherembodiment, the methods of the present invention provide that a singlecell suspension is incubated in an incubator further maintaining 6% CO₂.

In another embodiment, the methods of the present invention provide thata single cell suspension is incubated in an incubator furthermaintaining 60-100% humidity. In another embodiment, the methods of thepresent invention provide that a single cell suspension is incubated inan incubator further maintaining 70-100% humidity. In anotherembodiment, the methods of the present invention provide that a singlecell suspension is incubated in an incubator further maintaining 80-100%humidity. In another embodiment, the methods of the present inventionprovide that a single cell suspension is incubated in an incubatorfurther maintaining 90-100% humidity. In another embodiment, the methodsof the present invention provide that a single cell suspension isincubated in an incubator further maintaining 95-100% humidity.

In another embodiment, the methods of the present invention provide thata single cell suspension is plated at a final density of less than 1×10⁶cells/ml. In another embodiment, the methods of the present inventionprovide that a single cell suspension is plated at a final density ofless than 5×10⁵ cells/ml. In another embodiment, the methods of thepresent invention provide that a single cell suspension is plated at afinal density of less than 1×10⁵ cells/ml. In another embodiment, themethods of the present invention provide that a single cell suspensionis plated at a final density of less than 8×10⁴ cells/ml. In anotherembodiment, the methods of the present invention provide that a singlecell suspension is plated at a final density of about 6×10⁴ cells/ml(Example 1).

In another embodiment, the present invention provides a compositioncomprising disc stem cells. In another embodiment, the subject inventioncomprises a composition comprising a population of nucleus pulposuscells enriched for nucleus pulposus stem cells. In another embodiment,the composition further comprises an appropriate environment, such asthose described herein, wherein, a disc stem cell can be induced toproliferate and generate disc stem cells progeny. In another embodiment,the term environment in which disc stem cells progeny are placed, refersto the combination of external or extrinsic physical and/or chemicalconditions that affect and influence the growth and development of discstem cells. In another embodiment, the environment can be ex-vivo orin-vivo. In another embodiment, a disc scaffold can serve as an in-vivoenvironment that induces disc stem cells to generate progeny. In anotherembodiment, the environment is ex-vivo and comprises disc stem cellsplaced in cell culture medium in an incubator (Example 1).

In another embodiment, the present invention provides a compositioncomprising disc stem cells and media. In another embodiment, the mediais a serum free media. In another embodiment, the composition comprisingdisc stem cells further comprises Epidermal Growth Factor (EGF)supplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 1-10 ng/ml EGF supplementedto the media. In another embodiment, the composition comprising discstem cells further comprises 1-100 ng/ml EGF supplemented to the media.In another embodiment, the composition comprising disc stem cellsfurther comprises 20-50 ng/ml EGF supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises50-100 ng/ml EGF supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 5-15 ng/ml EGFsupplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 8-12 ng/ml EGF supplementedto the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises FGF supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises FibroblastGrowth Factor 2 (FGF2) supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises 1-100 ng/mlFGF2 supplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 20-50 ng/ml FGF2supplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 50-100 ng/ml FGF2supplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 5-15 ng/ml FGF2supplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 8-12 ng/ml FGF2supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises insulin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises1-100 μg/ml insulin supplemented to the media (Example 1). In anotherembodiment, the composition comprising disc stem cells further comprises20-50 μg/ml insulin supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises 50-100μg/ml insulin supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 5-15 μg/mlinsulin supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 8-12 μg/mlinsulin supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises progesterone supplemented to the media (Example 1). Inanother embodiment, the composition comprising disc stem cells furthercomprises 1-200 ng/ml progesterone supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises20-200 ng/ml progesterone supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises50-150 ng/ml progesterone supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises10-100 ng/ml progesterone supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises20-80 ng/ml progesterone supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises30-50 ng/ml progesterone supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises putrescine supplemented to the media (Example 1). Inanother embodiment, the composition comprising disc stem cells furthercomprises 1-800 ng/ml putrescine supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises1-100 ng/ml putrescine supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises 100-300ng/ml putrescine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 300-500 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 500-800 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 150-250 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 180-220 ng/mlputrescine supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises transferrin supplemented to the media (Example 1). Inanother embodiment, the composition comprising disc stem cells furthercomprises 1-400 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises1-100 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises100-200 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises200-400 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises20-150 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises80-200 ng/ml transferrin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises80-120 ng/ml transferrin supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises sodium selenite supplemented to the media (Example 1).In another embodiment, the composition comprising disc stem cellsfurther comprises 1-400 ng/ml sodium selenite supplemented to the media.In another embodiment, the composition comprising disc stem cellsfurther comprises 1-100 ng/ml sodium selenite supplemented to the media.In another embodiment, the composition comprising disc stem cellsfurther comprises 100-200 ng/ml sodium selenite supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 200-400 ng/ml sodium selenite supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 20-150 ng/ml sodium selenite supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 40-180 ng/ml sodium selenite supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 40-80 ng/ml sodium selenite supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises methylcellulose supplemented to the media (Example 1).In another embodiment, the composition comprising disc stem cellsfurther comprises 0.5-10% methylcellulose supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.5-3% methylcellulose supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises3-5% methylcellulose supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises 5-8%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 7-10%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 0.5-2.5%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 1-2.5%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 1.5-2.5%methylcellulose supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises an antibiotic supplemented to the media (Example 1).In another embodiment, the antibiotic supplemented to the media ispenicillin-streptomycin. In another embodiment, the compositioncomprising disc stem cells further comprises 1000-10000 U/mlpenicillin-streptomycin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises1000-3000 U/ml penicillin-streptomycin supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 3000-6000 U/ml penicillin-streptomycin supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 6000-10000 U/ml penicillin-streptomycin supplementedto the media. In another embodiment, the composition comprising discstem cells further comprises 3000-8000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, the compositioncomprising disc stem cells further comprises 4000-6000 U/mlpenicillin-streptomycin supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises5000 U/ml penicillin-streptomycin supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises KO serum replacer supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.5-30% KO serum replacer supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 5-30% knockout (KO) serum replacer supplemented to the media.In another embodiment, the composition comprising disc stem cellsfurther comprises 3-5% KO serum replacer supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 5-15% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises15-30% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises10-20% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises15-25% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises20% KO serum replacer supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises non-essential Amino Acids supplemented to the media.In another embodiment, the composition comprising disc stem cellsfurther comprises 0.1-10% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 0.1-1% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 1-5% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 5-10% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 15-30% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 0.5-1% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 0.8-1.2% non-essential Amino Acids supplemented to themedia. In another embodiment, the composition comprising disc stem cellsfurther comprises 1% non-essential Amino Acids supplemented to themedia.

In another embodiment, the composition comprising disc stem cellsfurther comprises L-glutamine supplemented to the media. In anotherembodiment, the composition comprising disc stem cells further comprises0.1-10 mM L-glutamine supplemented to the media. In another embodiment,the composition comprising disc stem cells further comprises 0.1-5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 5-10 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 5-8 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 0.5-2.5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 1.5-3 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 0.5-1.5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 0.8-1.2 mML-glutamine supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.01-1 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.01-0.5 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.5-1 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.5-0.8 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 00.5-0.25 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.15-0.3 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.05-0.15 mM b-mercaptoethanol supplemented to the media. Inanother embodiment, the composition comprising disc stem cells furthercomprises 0.08-0.12 mM b-mercaptoethanol supplemented to the media.

In another embodiment, the composition comprising disc stem cellsfurther comprises a media comprising Dulbecco's Modified Eagle's Medium(DMEM). In another embodiment, the composition comprising a discospherefurther comprises a media comprising DMEM/F12. In another embodiment,the composition comprising disc stem cells further comprises a mediacomprising Hamm's culture media. In another embodiment, the compositioncomprising disc stem cells further comprises a media comprisingHamm's/F12 culture media. In another embodiment, the compositioncomprising disc stem cells further comprises a media comprising ESGROComplete™ Accutase™. In another embodiment, ESGRO Complete™ Accutase™ isa cell detachment solution of proteolytic and collagenolytic enzymesqualified for use for the detachment of stem cells cultured inserum-free conditions with ESGRO Complete™ Clonal Grade Medium. Inanother embodiment, 1× Accutase™ enzymes in Dulbecco's PBS comprises 0.5mM EDTA•4Na and 3 mg/L Phenol. In another embodiment, the compositioncomprising disc stem cells further comprises a media comprising HEScGROhES cell medium (Chemicon Temecula, Calif.).

In another embodiment, the composition comprising disc stem cells isused for plating disc stem cells in ultra low attachment plates. Inanother embodiment, the composition comprising disc stem cells is usedfor plating disc stem cells in ultra low attachment plates precoatedwith an anti-adhesive substance. In another embodiment, theanti-adhesive substance is poly 2-hydroxyethyl methacrylate.

In another embodiment, the present invention provides an isolateddiscosphere. In another embodiment, a disc stem cell of the presentinvention gives rise to an isolated discosphere. In another embodiment,a discosphere of the present invention is the result of stem cellproliferation which gives rise to additional stem cells and progenitorcells. In another embodiment, a discosphere is formed as a result ofdisc stem proliferation.

In another embodiment, an isolated discosphere of the present inventioncomprises nucleus pulposus stem cells and nucleus pulposus progenitorcells arranged in a circular-spherical structure. In another embodiment,an isolated discosphere is a ball of cells in which a single disc stemcell gives rise to clones of itself (symmetric division) and toprogenitor cells. In another embodiment, a discosphere of the presentinvention is a free floating conglomerate of nucleus pulposus stem cellsand nucleus pulposus progenitor cells arranged in a circular-sphericalstructure. In another embodiment, a discosphere culture of the presentinvention comprises solitary free floating discospheres.

In another embodiment, the methods of the present invention provide thatisolating a discosphere of the present invention can be readilypreformed by one skilled in the art under a light microscope.

In another embodiment, the methods of the present invention provide thata single cell suspension is prepared for isolating a disc stem cell byplating and incubating a disc stem cell in a serum free media. Inanother embodiment, the methods of the present invention provide that asingle cell suspension is prepared for producing a discosphere byplating and incubating a disc stem cell in a serum free media.

In another embodiment, the present invention provides a method ofproducing a discosphere, comprising the step of growing a culture ofnucleus pulposus cells in a serum free media, thereby producing adiscosphere. In another embodiment, the present invention provides thatgrowing a primary culture of nucleus pulposus cells in a serum freemedia results in selecting nucleus pulposus stem cells. In anotherembodiment, the remaining isolated culture of nucleus pulposus stemcells gives rise to discospheres of the present invention. In anotherembodiment, the remaining enriched culture of nucleus pulposus stemcells gives rise to discospheres of the present invention. In anotherembodiment, discospheres according to the methods of the presentinvention grow in the compositions of the present invention. In anotherembodiment, discospheres according to the methods of the presentinvention grow in the supplemented media of the present invention. Inanother embodiment, discospheres according to the methods of the presentinvention grow under conditions which do not permit cell-substrateadhesion. In another embodiment, conditions which do not permitcell-substrate adhesion comprise for example the addition of about0.2-2% methylcellulose to the cell culture media of the presentinvention.

In another embodiment, the present invention provides a compositioncomprising a discosphere. In another embodiment, a composition of thepresent invention comprises a single discosphere. In another embodiment,a composition of the present invention comprises at least 1×10²discospheres. In another embodiment, a composition of the presentinvention comprises at least 1×10³ discospheres. In another embodiment,a composition of the present invention comprises at least 1×10⁴discospheres. In another embodiment, a composition of the presentinvention comprises at least 1×10⁵ discospheres. In another embodiment,a composition of the present invention comprises at least 1×10⁶discospheres.

In another embodiment, the composition of the present inventioncomprises a discosphere and media. In another embodiment, the media is aserum free media. In another embodiment, the composition comprising adiscosphere further comprises EGF supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises1-10 ng/ml EGF supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 1-100 ng/ml EGFsupplemented to the media. In another embodiment, the compositioncomprising a discosphere further comprises 20-50 ng/ml EGF supplementedto the media. In another embodiment, the composition comprising adiscosphere further comprises 50-100 ng/ml EGF supplemented to themedia. In another embodiment, the composition comprising a discospherefurther comprises 5-15 ng/ml EGF supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises8-12 ng/ml EGF supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises FGF supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises FGF2 supplementedto the media. In another embodiment, the composition comprising adiscosphere further comprises 1-100 ng/ml FGF2 supplemented to themedia. In another embodiment, the composition comprising a discospherefurther comprises 20-50 ng/ml FGF2 supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises50-100 ng/ml FGF2 supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 5-15 ng/ml FGF2supplemented to the media. In another embodiment, the compositioncomprising a discosphere further comprises 8-12 ng/ml FGF2 supplementedto the media.

In another embodiment, the composition comprising a discosphere furthercomprises insulin supplemented to the media (Example 2 and materials andmethods). In another embodiment, the composition comprising adiscosphere further comprises 1-100 μg/ml insulin supplemented to themedia. In another embodiment, the composition comprising a discospherefurther comprises 20-50 μg/ml insulin supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 50-100 μg/ml insulin supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises5-15 μg/ml insulin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 3-7 μg/ml insulinsupplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises progesterone supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 1-200 ng/mlprogesterone supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 20-200 ng/mlprogesterone supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 50-150 ng/mlprogesterone supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 10-100 ng/mlprogesterone supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 20-80 ng/mlprogesterone supplemented to the media. In another embodiment, thecomposition comprising disc stem cells further comprises 15-25 ng/mlprogesterone supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises putrescine supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 1-800 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 1-100 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 100-300 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 300-500 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 500-800 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 150-250 ng/mlputrescine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 140-160 ng/mlputrescine supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises transferrin supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 1-400 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 1-100 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 100-200 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 200-400 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 20-150 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 80-200 ng/mltransferrin supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 30-70 ng/mltransferrin supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises1-400 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises1-100 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises100-200 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises200-400 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises20-150 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises40-180 ng/ml sodium selenite supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises20-40 ng/ml sodium selenite supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises methylcellulose supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.5-10% methylcellulose supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.5-3% methylcellulose supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 3-5%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 5-8%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 7-10%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.5-2.5%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 1-2.5%methylcellulose supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.6-1%methylcellulose supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises an antibiotic supplemented to the media. In anotherembodiment, the antibiotic supplemented to the media ispenicillin-streptomycin. In another embodiment, the compositioncomprising a discosphere further comprises 1000-10000 U/mlpenicillin-streptomycin supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises1000-3000 U/ml penicillin-streptomycin supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 3000-6000 U/ml penicillin-streptomycin supplemented to themedia. In another embodiment, the composition comprising a discospherefurther comprises 6000-10000 U/ml penicillin-streptomycin supplementedto the media. In another embodiment, the composition comprising adiscosphere further comprises 3000-8000 U/ml penicillin-streptomycinsupplemented to the media. In another embodiment, the compositioncomprising a discosphere further comprises 4000-6000 U/mlpenicillin-streptomycin supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises5000 U/ml penicillin-streptomycin supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.5-30% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises5-30% KO serum replacer supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises3-5% KO serum replacer supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 5-15% KOserum replacer supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 15-30% KO serumreplacer supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 10-20% KO serumreplacer supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 15-25% KO serumreplacer supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 20% KO serumreplacer supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 0.1-10% non-essential Amino Acids supplemented to the media.In another embodiment, the composition comprising a discosphere furthercomprises 0.1-1% non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 1-5% non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 5-10% non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 15-30% non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 0.5-1% non-essential Amino Acids supplemented to the media. Inanother embodiment, the composition comprising a discosphere furthercomprises 0.8-1.2% non-essential Amino Acids supplemented to the media.In another embodiment, the composition comprising a discosphere furthercomprises 1% non-essential Amino Acids supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises L-glutamine supplemented to the media. In another embodiment,the composition comprising a discosphere further comprises 0.1-10 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.1-5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 5-10 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 5-8 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.5-2.5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 1.5-3 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.5-1.5 mML-glutamine supplemented to the media. In another embodiment, thecomposition comprising a discosphere further comprises 0.8-1.2 mML-glutamine supplemented to the media.

In another embodiment, the composition comprising a discosphere furthercomprises b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.01-1 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.01-0.5 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.5-1 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.5-0.8 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises00.5-0.25 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.15-0.3 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.05-0.15 mM b-mercaptoethanol supplemented to the media. In anotherembodiment, the composition comprising a discosphere further comprises0.08-0.12 mM b-mercaptoethanol supplemented to the media.

In another embodiment, the composition comprising a discospherecomprises a media comprising Dulbecco's Modified Eagle's Medium (DMEM).In another embodiment, the composition comprising a discospherecomprises a media further comprising DMEM/F12. In another embodiment,the composition comprising a discosphere comprises a media furthercomprising ESGRO Complete™ Accutase™. In another embodiment, ESGROComplete™ Accutase™ is a cell detachment solution of proteolytic andcollagenolytic enzymes qualified for use for the detachment of stemcells cultured in serum-free conditions with ESGRO Complete™ ClonalGrade Medium. In another embodiment, 1× Accutase™ enzymes in Dulbecco'sPBS comprises 0.5 mM EDTA•4Na and 3 mg/L Phenol. In another embodiment,the composition comprising a discosphere comprises a media comprisingHEScGRO hES cell medium (Chemicon Temecula, Calif.).

In another embodiment, the present invention provides that discospheresobtained by the methods of the present invention are further expanded.In another embodiment, the present invention provides that discospheresare dissociated by incubation at 37° C. in DMEM/F12 medium supplementedwith collagenase. In another embodiment, the present invention providesthat the dissociated cells are expanded by replating the same intomethylcellulose-based medium.

In another embodiment, the methods of the present invention provide thatthe medium is supplemented with 8-20% fetal bovine serum (FBS) whengrowing a disc tissue. In another embodiment, the methods of the presentinvention provide that the medium is supplemented with 8-20% fetalbovine serum (FBS) when growing a disc tissue in a scaffold. In anotherembodiment, the medium comprises 30-70% media derived from cultures ofprimary human foreskin fibroblasts, or a combination thereof. In anotherembodiment, the methods of the present invention provide that the mediumis free of serum when growing disc stem cells, disc progenitor cells, ora combination thereof. In another embodiment, the methods of the presentinvention provide that the medium is free of a serum replacer whengrowing disc stem cells, disc progenitor cells, or a combinationthereof. In another embodiment, the methods of the present inventionprovide that the medium is free of FBS when growing disc stem cells. Inanother embodiment, the methods of the present invention provide thatthe medium is free of FBS when growing nucleus pulposus stem cells. Inanother embodiment, the methods of the present invention provide thatthe medium is free of FBS when growing progenitor cells. In anotherembodiment, the methods of the present invention provide that the mediumis free of FBS when growing nucleus pulposus progenitor cells.

In another embodiment, the methods of the present invention provide thatthe medium is free of serum or a serum replacer when isolating disc stemcells or disc progenitor cells. In another embodiment, the methods ofthe present invention provide that the medium is free of serum or aserum replacer when enriching a heterogeneous population of cells fordisc stem cells, disc progenitor cells, or a combination thereof. Inanother embodiment, the methods of the present invention provide thatthe medium is free of serum or a serum replacer when growingdiscospheres comprising disc stem cells, disc progenitor cells, or acombination thereof. In another embodiment, the methods of the presentinvention provide that the medium is free of serum or a serum replacerwhen expanding a culture comprising disc stem cells, disc progenitorcells, or a combination thereof. In another embodiment, the methods ofthe present invention provide that the medium is free of serum or aserum replacer when expanding a culture enriched for disc stem cells,disc progenitor cells, or a combination thereof.

In another embodiment, the present invention provides a disc replacementdevice comprising nucleus pulposus cells. In another embodiment, thepresent invention provides an artificial disc comprising nucleuspulposus cells. In another embodiment, the disc replacement device is anintervertebral disc replacement device. In another embodiment, anintervertebral disc is located between the concave articular surfaces ofthe adjacent vertebral body endplates. In another embodiment, the discreplacement device of the present invention permits movements such asflexion, extension, lateral flexion, and rotation. In anotherembodiment, the disc replacement device of the present invention is usedto repair and/or replace injured or damaged intervertebral discs. Inanother embodiment, the disc replacement device of the present inventionprovides a prosthetic disc that combines both stability to support thehigh loads, of the patient's vertebrae and flexibility to provide thepatient with sufficient mobility and proper spinal column loaddistribution.

In another embodiment, the disc replacement device comprises a discscaffold. In another embodiment, the scaffold comprises a shape memoryalloy. In another embodiment, a shape memory alloy may be deformedduring its martensitic phase, but will regain its original shape when itis heated above a certain temperature, such as an austenite phasetemperature. In another embodiment, a shape memory alloy of the presentinvention exhibits a superelastic property, thereby able to absorb largedeformations without damaging its structure.

In another embodiment, the disc replacement device comprises a rigidbody that fits between the vertebrates with a protuberance extendingfrom a vertebral contacting surface and extends into the vertebral body.In another embodiment, the disc replacement device comprises a discarthroplasty device for replacement of the spinal disk. In anotherembodiment, the disc replacement device comprises a ball-and-socket toenable rotation. In another embodiment, the disc replacement devicecomprises an intermediate layer allowing for movement between the upperjoint piece and the lower joint piece.

In another embodiment, the disc replacement device comprises twoendplates that are anchored to the top and bottom surfaces of the spinalbones. In another embodiment, the disc replacement device comprises twometal endplates that are anchored to the top and bottom surfaces of thespinal bones. In another embodiment, the metal is cobalt-chrome alloy.In another embodiment, the endplates are coated with nucleus pulposuscell adhesion molecules. In another embodiment, the endplates are coatedwith molecules promoting nucleus pulposus cell growth.

In another embodiment, the disc replacement device comprises ceramics.In another embodiment, the disc replacement device comprises injectablefluids. In another embodiment, the disc replacement device compriseshydrogels. In another embodiment, the disc replacement device comprisesa hydrogel core in a flexible, inelastic, woven polyethylene jacket. Inanother embodiment, the disc replacement device comprises a polyvinylalcohol material. In another embodiment, the disc replacement devicecomprises inflatables. In another embodiment, the disc replacementdevice comprises elastic coils. In another embodiment, the discreplacement device comprises an elongated elastic memory-coiling spiral.In another embodiment, the elongated elastic memory-coiling spiral ismade of polycarbonate urethane. In another embodiment, the discreplacement device comprises a one-piece convex surfaced ceramic ormetal implant that anchors to the inferior vertebral body as ahemiarthroplasty. In another embodiment, the disc replacement devicecomprises a balloon-like implant made of polyurethane. In anotherembodiment, the disc replacement device comprises a protein hydrogel. Inanother embodiment, the disc replacement device comprises athermopolymer.

In another embodiment, the disc scaffold comprises an ECM component. Inanother embodiment, the ECM component is a structural protein. Inanother embodiment, the disc scaffold comprises collagen. In anotherembodiment, the structural protein is elastin. In some embodiments, theECM component is a specialized protein. In another embodiment, thespecialized protein is fibrillin. In another embodiment, the specializedprotein is fibronectin. In another embodiment, the specialized proteinis laminin. In some embodiments, the ECM component is a proteoglycan. Inone embodiment, proteoglycans are composed of a protein core to which isattached long chains of repeating disaccharide units termed ofglycosaminoglycans (GAGs) forming extremely complex high molecularweight component.

In another embodiment, collagen is collagen type I. In anotherembodiment, collagen type I comprises [a1(I)]2[a(I)] chains. In anotherembodiment, collagen type I is derived from skin, tendon, or bone.

In another embodiment, collagen is collagen type II. In anotherembodiment, collagen type II comprises [a1(II)]3 chains. In anotherembodiment, collagen type II is derived from cartilage or vitreoushumor. In another embodiment, type II collagen fibrils are cross-linkedto proteoglycans in the matrix by type IX collagen.

In another embodiment, collagen is collagen type III. In anotherembodiment, collagen type III comprises [a1(III)]3 chains. In anotherembodiment, collagen type III is derived from skin or muscle, and isfrequently found with type I.

In another embodiment, collagen is collagen type IV. In anotherembodiment, collagen type IV comprises [a1(IV)2[a2(IV)] chains. Inanother embodiment, collagen type IV is derived from basal lamina.

In another embodiment, collagen is collagen type V. In anotherembodiment, collagen type V comprises [a1(V)][a2(V)][a3(V)] chains. Inanother embodiment, collagen type V is derived from an interstitialtissue associated with type I collagen.

In another embodiment, collagen is collagen type VI. In anotherembodiment, collagen type VI comprises [a1(VI)] [a2(VI)] [a3(VI)]chains. In another embodiment, collagen type VI is derived from aninterstitial tissue associated with type I collagen. In anotherembodiment, type VI collagen consists of relatively short triple-helicalregions about 60 nm long separated by globular domains about 40 nm long.In some embodiments, fibrils of pure type VI collagen form a structuresimilar to beads on a string.

In one embodiment, collagen is collagen type VII. In one embodiment,collagen type VII comprises [a1(VII)]3 chains. In another embodiment,collagen type VII is derived from epithelia.

In another embodiment, collagen is collagen type VIII. In anotherembodiment, collagen type VIII comprises [a1(VIII)]3 chains. In anotherembodiment, collagen type VII is derived from endothelial cells.

In another embodiment, collagen is collagen type IX. In anotherembodiment, collagen type IX comprises [a1(IX)][a2(IX)][a3(IX)] chains.In another embodiment, collagen type IX is derived from cartilageassociated with type II collagen.

In another embodiment, collagen is collagen type X. In anotherembodiment, collagen type X comprises [a1(X)]3 chains. In anotherembodiment, collagen type X is derived from hypertrophic andmineralizing cartilage.

In another embodiment, collagen is collagen type XI. In anotherembodiment, collagen type XI comprises [a1(XI)][a2(XI)][a3(XI)] chains.In another embodiment, collagen type XI is derived from cartilage.

In another embodiment, collagen is collagen type XII. In anotherembodiment, collagen type XII comprises a1(XII) chains. In anotherembodiment, collagen type XII is derived from sites wherein types I andIII collagens are present.

In another embodiment, type I collagen molecules pack togetherside-by-side, forming fibrils with a diameter of 50-200 nm. In someembodiments, fibrils, adjacent collagen molecules are displaced from oneanother by 67 nm, about another-quarter of their length. In someembodiments, collagens types I, II, III, and V form rodlike triplehelices to via side-by-side interactions.

In another embodiment, the collagen of the present invention is derivedfrom cows. In another embodiment, collagen of the present invention isderived from patient's own fat or hyaluronic acid.

In another embodiment, collagen is a collagen-like substance which hasbeen modified by dissolving collagen in water and modifying the thuslydissolved collagen to render its surface charge effectively morepositive than prior to modification. In another embodiment, thismaterial is well known and is disclosed, e.g., in U.S. Pat. No.4,238,480. In another embodiment, modified collagen is freeze-dried toform a solid mass of gelatin. In some embodiments, the mass of gelatinmay be formed in the shape of a rod, strip, film or flake.

In another embodiment, other forms of collagen which are suitable foruse in the present invention include Semed F, a collagen preparationmanufactured in native fiber form without any chemical or enzymaticmodifications, and Semed S, a lyophilized collagen powder extracted fromfresh bovine hides. In another embodiment, the Semed F material is aType I collagen (greater than 95%), while the Semed S is a mixture ofType I and Type III collagen macro-molecules in which the shape anddimension of tropocollagen in its natural helical orientation isretained.

In another embodiment, the concentration of the collagen in the liquidwhich is to be freeze-dried can range from 0.5-10% and preferably 1-5%,with the lower concentrations forming less dense or discontinuoussolids. In another embodiment, at lower concentrations of 0.5 to 1%, theSemed F forms a structure which approximates dense cobwebs.

In another embodiment, native collagen film, wherein the film strengthis preserved and the triple-helix structure of the collagen polymer ismaintained intact, can also be used, either alone or with a plasticizerincorporated therewith.

In another embodiment, gelatin or other water soluble forms of collagenare utilized. In another embodiment, soluble forms of collagen willreadily polymerize at body temperatures to form a stable subcutaneousgel. In another embodiment, when soluble forms of collagen are implantedinto the body, the polymerized material will become rapidly populated bynucleus pulposus cells implanted therein and host fibroblasts. In someembodiments, the material becomes vascularized and can remainhistologically stable. In another embodiment, the material becomesvascularized and can remain histologically stable for at least 4 months.In another embodiment, the material becomes vascularized and can remainhistologically stable for at least 6 months. In another embodiment, thematerial becomes vascularized and can remain histologically stable forat least 8 months. In another embodiment, the material becomesvascularized and can remain histologically stable for at least 10months. In another embodiment, the material becomes vascularized and canremain histologically stable for at least 12 months. In anotherembodiment, the material becomes vascularized and can remainhistologically stable for at least 15 months. In another embodiment, thematerial becomes vascularized and can remain histologically stable forat least 18 months.

In another embodiment, the present invention provides mixtures of thevarious types of collagen of the invention to obtain the most desirablefeatures of each grade.

In another embodiment, fibronectins are dimers of 2 similar peptides. Inanother embodiment, each chain of a fibronectins is 60-70 nm long and2-3 nm thick. In another embodiment, fibronectins contain at least 6tightly folded domains each with a high affinity for a differentsubstrate such as heparan sulfate, collagen (separate domains for typesI, II and III collagens), and fibrin and cell-surface receptors.

In another embodiment, laminin molecule is a heterotrimer assembled fromα, β, and γ-chains. In some embodiments, laminins form independentnetworks and are associated with type IV collagen networks via entactin,and perlecan. In some embodiments, laminins contribute to cellviability, attachment, and differentiation, cell shape and movement,maintenance of tissue phenotype, and promotion of tissue survival.

In another embodiment, proteoglycans comprise chondroitin sulfate anddermatan sulfate chains. In another embodiment, proteoglycans compriseheparin and heparan sulfate chains. In another embodiment, proteoglycanscomprise keratan sulfate chains. In another embodiment, proteoglycansare aggrecans, the major proteoglycan in cartilage. In anotherembodiment, proteoglycans are versican, present in many adult tissuesincluding blood vessels and skin. In another embodiment, proteoglycansare small leucine rich repeat proteoglycans (SLRPs). In anotherembodiment, SLRPs include decorin, biglycan, fibromodulin, and lumican.

In another embodiment, the extracellular matrix components aremorselized. In another embodiment, morselization of the extracellularmatrix proteins increases the surface area for nucleus pulposus cellsattachment. In another embodiment, morselization of the extracellularmatrix proteins increases the surface area for discospheres attachment.In another embodiment, morselization of the extracellular matrixproteins increases the surface area for disc stem cells attachment. Inanother embodiment, morselization of the extracellular matrix proteinsincreases the surface area for disc progenitor cells attachment. Inanother embodiment, morselization of the extracellular matrix proteinsincreases the surface area thus aiding diffusion of nutrients and wasteproducts to the implant and from the implant. In another embodiment,morselization of the extracellular matrix proteins allows theintroduction of nucleus pulposus cells into the disc scaffold through aneedle or a small cannula. In another embodiment, morselization of theextracellular matrix proteins allows the introduction of discospheresinto the disc scaffold through a needle or a small cannula. In anotherembodiment, small holes could be drilled into the disc scaffold for cellattachment.

In another embodiment, the present invention provides that the discscaffold is obtained from an animal or human. In another embodiment, thepresent invention provides that the disc scaffold is an intervertebraldisc with the vertebral endplates left intact. In another embodiment,the present invention provides that the disc scaffold is a rabbitintervertebral disc with the vertebral endplates left intact (Example3). In another embodiment, the present invention provides that the discscaffold is a dog intervertebral disc with the vertebral endplates leftintact. In another embodiment, the present invention provides that thedisc scaffold is a horse intervertebral disc with the vertebralendplates left intact. In another embodiment, the present inventionprovides that the disc scaffold is a monkey intervertebral disc with thevertebral endplates left intact. In another embodiment, the presentinvention provides that the disc scaffold is a pig intervertebral discwith the vertebral endplates left intact. In another embodiment, thepresent invention provides that the disc scaffold is a cowintervertebral disc with the vertebral endplates left intact. In anotherembodiment, the present invention provides that the disc scaffoldcomprising collagen further comprises additional material such asceramics or metals.

In another embodiment, the present invention provides that the discreplacement device comprises nucleus pulposus cells. In anotherembodiment, the present invention provides that the disc replacementdevice comprises human nucleus pulposus cells. In another embodiment,the present invention provides that the disc replacement devicecomprises nucleus pulposus stem cells. In another embodiment, thepresent invention provides that the disc replacement device comprisesnucleus pulposus progenitor cells. In another embodiment, the presentinvention provides that the disc replacement device comprisesdiscospheres of the present invention.

In another embodiment, the disc replacement device further comprisesmedia. In another embodiment, the media comprises cell culture media ofthe present invention (Example 3).

In another embodiment, the present invention provides a method ofproducing an artificial disc, comprising the step of growingdiscospheres in a disc scaffold. In another embodiment, the presentinvention provides a method of producing an intervertebral discreplacement device, comprising the step of growing discospheres in adisc scaffold. In another embodiment, discospheres are administered ontoa disc scaffold. In another embodiment, discospheres are administeredinto a layer comprising collagen in the disc scaffold. In anotherembodiment, discospheres are administered onto a layer comprisingcollagen in the disc scaffold. In another embodiment, discospheres areinjected into a disc scaffold (Example 4). In another embodiment,discospheres are injected onto a disc scaffold. In another embodiment,discospheres are injected into a layer comprising collagen in the discscaffold. In another embodiment, discospheres are injected onto a layercomprising collagen in the disc scaffold. In another embodiment, thediscospheres of the present invention are applied or injected into oronto the disc scaffold together with a composition of the presentinvention. In another embodiment, the discospheres of the presentinvention are applied or injected into or onto the disc scaffoldtogether with a DMEM/F12 medium supplemented with 10% FCS.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprising the stepof growing nucleus pulposus cells in a disc scaffold. In anotherembodiment, the present invention provides a method of producing aspinal disc tissue, comprising the step of growing discospheres in adisc scaffold, thereby producing a spinal disc tissue. In anotherembodiment, a spinal disc tissue of the present invention comprises adisc scaffold of the present invention. In another embodiment, a spinaldisc tissue of the present invention comprises nucleus pulposus cells ofthe present invention. In another embodiment, a spinal disc tissue ofthe present invention comprises a disc scaffold of the presentinvention. In another embodiment, a spinal disc tissue of the presentinvention comprises nucleus pulposus cells of the present inventiongrown on a disc scaffold of the present invention. In anotherembodiment, a spinal disc tissue of the present invention comprises adisc scaffold of the present invention. In another embodiment, a spinaldisc tissue of the present invention comprises matured nucleus pulposuscells derived from discospheres of the present invention attached to adisc scaffold of the present invention. In another embodiment, a spinaldisc tissue of the present invention comprises matured nucleus pulposuscells derived from disc stem cells of the present invention attached toa disc scaffold of the present invention. In another embodiment, aspinal disc tissue of the present invention comprises fibroblasts andmatured nucleus pulposus cells derived from discospheres of the presentinvention attached to a disc scaffold of the present invention.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprising coatingthe disc scaffold of the present invention with nucleus pulposus cellsgrowth factors. In another embodiment, the present invention provides amethod of producing an intervertebral disc replacement device,comprising coating the disc scaffold of the present invention withnucleus pulposus cells adhesion factors. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising coating the disc scaffold of the presentinvention with nucleus pulposus cells differentiation factors. Inanother embodiment, the present invention provides a method of producingan intervertebral disc replacement device, comprising placing the discscaffold of the present invention in a media comprising nucleus pulposuscells growth factors, adhesion factors, and differentiation factors. Inanother embodiment, the present invention provides a method of producingan intervertebral disc replacement device, comprising placing the discscaffold of the present invention in a cell culture media comprisingnucleus pulposus cells growth factors, adhesion factors, anddifferentiation factors. In another embodiment, the present inventionprovides a method of producing an intervertebral disc replacementdevice, comprising placing the disc scaffold of the present invention ina media comprising DMEM/F12 medium. In another embodiment, the presentinvention provides a method of producing an intervertebral discreplacement device, comprising placing the disc scaffold of the presentinvention in a media comprising DMEM/F12 medium and 10% fetal calf serum(FCS) (Example 3).

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprisingincubating the disc scaffold of the present invention in a media of theinvention at 35-42° C. In another embodiment, the present inventionprovides a method of producing an intervertebral disc replacementdevice, comprising incubating the disc scaffold of the present inventionin a media of the invention at 36-38° C. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising incubating the disc scaffold of thepresent invention in a media of the invention at 37° C.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprisingincubating the disc scaffold of the present invention in a media of theinvention while maintaining 4-10% CO₂. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising incubating the disc scaffold of thepresent invention in a media of the invention while maintaining 4-8%CO₂. In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprisingincubating the disc scaffold of the present invention in a media of theinvention while maintaining 5% CO₂.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprisingincubating the disc scaffold of the present invention in a media of theinvention for 2-12 hours in an incubator. In another embodiment, thepresent invention provides a method of producing an intervertebral discreplacement device, comprising incubating the disc scaffold of thepresent invention in a media of the invention for 3-10 hours in anincubator. In another embodiment, the present invention provides amethod of producing an intervertebral disc replacement device,comprising incubating the disc scaffold of the present invention in amedia of the invention for 6-10 hours in an incubator. In anotherembodiment, the present invention provides a method of producing anintervertebral disc replacement device, comprising incubating the discscaffold of the present invention in a media of the invention for 8hours in an incubator.

In another embodiment, the present invention provides that incubatingthe disc scaffold of the present invention in a media of the inventionin an incubator comprises a step for preparing the disc scaffold beforenucleus pulposus cells are applied into or onto the disc scaffold. Inanother embodiment, the present invention provides that incubating thedisc scaffold of the present invention in a media of the invention in anincubator enable nucleus pulposus cells of the invention to adhere, growand differentiate on the disc scaffold. In another embodiment, thepresent invention provides that incubating the disc scaffold of thepresent invention in a media of the invention in an incubator enablediscospheres of the invention to adhere, grow and differentiate on thedisc scaffold. In another embodiment, the present invention providesthat incubating the disc progenitor cells of the present invention in amedia of the invention in an incubator enable nucleus pulposus cells ofthe invention to adhere, grow and differentiate on the disc scaffold. Inanother embodiment, the present invention provides that incubating thedisc scaffold of the present invention in a media of the invention in anincubator enable disc stem cells of the invention to adhere, grow anddifferentiate on the disc scaffold. In another embodiment, the presentinvention provides that incubating the disc scaffold of the presentinvention in a media of the invention in an incubator enable nucleuspulposus cells, disc stem cells, disc progenitor cells, discospheres, orany combination thereof to adhere, grow and differentiate on the discscaffold.

In another embodiment, the present invention provides that autograftnucleus pulposus cells are harvested, cultured, and injected to thecenter of a disc scaffold (Example 4). In another embodiment, thepresent invention provides that allograft nucleus pulposus cells areharvested, cultured, and injected to the center of a disc scaffold. Inanother embodiment, the present invention provides that xenograftnucleus pulposus cells are harvested, cultured, and injected to thecenter of a disc scaffold.

In another embodiment, the present invention provides that nucleuspulposus stem cells, nucleus pulposus progenitor cells, discospheres, ora combination thereof are implanted into the disc scaffold to form aliving nucleus pulposus. In another embodiment, nucleus pulposus stemcells, nucleus pulposus progenitor cells, discospheres, or a combinationthereof obtained from a cell culture are implanted into the discscaffold to form a living nucleus pulposus.

In another embodiment, disc stem cells are administered onto a discscaffold. In another embodiment, disc stem cells are administered into alayer comprising collagen in the disc scaffold. In another embodiment,disc stem cells are administered onto a layer comprising collagen in thedisc scaffold. In another embodiment, disc stem cells are injected intoa disc scaffold. In another embodiment, disc stem cells are injectedonto a disc scaffold. In another embodiment, disc stem cells areinjected into a layer comprising collagen in the disc scaffold. Inanother embodiment, disc stem cells are injected onto a layer comprisingcollagen in the disc scaffold. In another embodiment, the disc stemcells of the present invention are applied or injected into or onto thedisc scaffold together with a composition of the present invention. Inanother embodiment, the disc stem cells of the present invention areapplied or injected into or onto the disc scaffold together with aDMEM/F12 medium with 10% FCS.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprising the stepof growing nucleus pulposus primary cells in a disc scaffold. In anotherembodiment, disc primary cells are administered onto a disc scaffold. Inanother embodiment, disc primary cells are administered into a layercomprising collagen in the disc scaffold. In another embodiment, discprimary cells are administered onto a layer comprising collagen in thedisc scaffold. In another embodiment, disc primary cells are injectedinto a disc scaffold. In another embodiment, disc primary cells areinjected onto a disc scaffold. In another embodiment, disc primary cellsare injected into a layer comprising collagen in the disc scaffold. Inanother embodiment, disc primary cells are injected onto a layercomprising collagen in the disc scaffold. In another embodiment, thedisc primary cells of the present invention are applied or injected intoor onto the disc scaffold together with a composition of the presentinvention. In another embodiment, the disc primary cells of the presentinvention are applied or injected into or onto the disc scaffoldtogether with a DMEM/F12 medium with 10% FCS.

In another embodiment, the present invention provides a method ofproducing an intervertebral disc replacement device, comprising the stepof collecting the discospheres, disc stem cells, disc progenitor cells,or a mixture thereof from cell culture media of the present invention bymethods known to a person with skill in the art and placing the cells inDMEM/F12. In another embodiment, the present invention provides thatdiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof are first washed free of cell-substrate adhesion inhibitoryfactor. In another embodiment, the present invention provides thatdiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof are first washed free of methylcellulose.

In another embodiment, the present invention provides that washeddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof substantially free of cell-substrate adhesion inhibitory factorsare placed in a cell culture media. In another embodiment, the presentinvention provides that washed discospheres, disc stem cells, discprogenitor cells, or a mixture thereof substantially free ofcell-substrate adhesion inhibitory factors are placed in DMEM. Inanother embodiment, the present invention provides that washeddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof substantially free of cell-substrate adhesion inhibitory factorsare placed in DMEM/F12. In another embodiment, the present inventionprovides that washed discospheres, disc stem cells, disc progenitorcells, or a mixture thereof substantially free of cell-substrateadhesion inhibitory factors are placed in DMEM/F12 comprising serum.

In another embodiment, discospheres, disc stem cells, disc progenitorcells, or a mixture thereof are introduced to the disc scaffold in acell culture media of the invention. In another embodiment,discospheres, disc stem cells, disc progenitor cells, or a mixturethereof are introduced to the disc scaffold in a cell culture mediacomprising recombinant generated morphogenetic proteins. In anotherembodiment, discospheres, disc stem cells, disc progenitor cells, or amixture thereof are introduced to the disc scaffold in a cell culturemedia comprising PDGF. In another embodiment, discospheres, disc stemcells, disc progenitor cells, or a mixture thereof are introduced to thedisc scaffold in a cell culture media comprising TGF-β. In anotherembodiment, discospheres, disc stem cells, disc progenitor cells, or amixture thereof are introduced to the disc scaffold in a cell culturemedia comprising EGF/TGF-α. In another embodiment, discospheres, discstem cells, disc progenitor cells, or a mixture thereof are introducedto the disc scaffold in a cell culture media comprising IGF-I. Inanother embodiment, discospheres, disc stem cells, disc progenitorcells, or a mixture thereof are introduced to the disc scaffold in acell culture media comprising βFGF. In another embodiment, discospheres,disc stem cells, disc progenitor cells, or a mixture thereof areintroduced to the disc scaffold in a cell culture media comprisinghydrogels. In another embodiment, discospheres, disc stem cells, discprogenitor cells, or a mixture thereof are introduced to the discscaffold in a cell culture media comprising absorbable or non-resorbablesynthetic or natural polymers such as but not limited to collagen,fibrin, polyglycolic acid, polylactic acid, or polytetrafluoroethylene.In another embodiment, discospheres, disc stem cells, disc progenitorcells, or a mixture thereof are introduced to the disc scaffold in acell culture media comprising antibiotics. In another embodiment,discospheres, disc stem cells, disc progenitor cells, or a mixturethereof are introduced to the disc scaffold in a cell culture mediacomprising anti-inflammatory medication. In another embodiment,discospheres, disc stem cells, disc progenitor cells, or a mixturethereof are introduced to the disc scaffold in a cell culture mediacomprising immunosuppressive medications.

In another embodiment, the present invention provides that the collagenfibers of the annulus fibrosis are arranged in 5-50 layers or lamella.In another embodiment, the present invention provides that the collagenfibers of the annulus fibrosis are arranged in 10-40 layers or lamella.In another embodiment, the present invention provides that the collagenfibers of the annulus fibrosis are arranged in 20-30 layers or lamella.

In another embodiment, the present invention provides that the fibers ofthe lamella alternate direction between layers. In another embodiment,the present invention provides that a blunt tipped needle or cannulacould be forced through the annulus. In another embodiment, the presentinvention provides that upon withdraw of the needle, after injecting thetransplanted nucleus pulposus ceels or discospheres, the separatedfibers of the lamella would return to their normal position, sealing theannulus. In another embodiment, the present invention provides that theneedle would be inserted into the anterior or lateral portion of thedisc scaffold. In another embodiment, the present invention providesthat those skilled in the art will realize that the needle could bedirected into the lateral portion of the disc percutaneously withfluoroscopic guidance and into the anterior portion of the disclaparoscopically.

In another embodiment, the present invention provides that the recipientof the nucleus pulposus cells of the present invention is the donor. Inanother embodiment, the present invention provides that the recipient ofthe nucleus pulposus cells of the present invention may function atleast in part as a donor. In another embodiment, the present inventionprovides that the donor of nucleus pulposus cells of the presentinvention is a single donor. In another embodiment, the presentinvention provides that multiple donors provide nucleus pulposus cellsof the present invention to a single recipient. In another embodiment,the present invention provides that multiple donors provide nucleuspulposus cells of the present invention to multiple recipients. Inanother embodiment, the present invention provides that fetal sourcesare used. In another embodiment, the present invention provides that thedonor or donors of the nucleus pulposus cells of the present inventionis or are preferably having a familial relationship to the recipient inorder to minimize or avoid immunosuppression. In another embodiment, thepresent invention provides that the donor or donors of the nucleuspulposus cells of the present invention is or are preferably having afamilial relationship to the recipient in order to minimize or avoid theneed for immunosuppressive substances. In another embodiment, thepresent invention provides guidelines for tissue procurement includingsurgical techniques of removal, number of hours between death of thedonor and tissue procurement, and testing of the donor for infectiousdisease, are well known to one of skill in the art.

In another embodiment, the present invention provides that nucleuspulposus cells injected into or onto the disc scaffold depositextracellular matrix components. In another embodiment, the presentinvention provides that discospheres injected into or onto the discscaffold deposit extracellular matrix components of the disc. In anotherembodiment, the present invention provides that these extracellularmatrix components shape the discs' subsequent physiological functions.In another embodiment, the present invention provides that theseextracellular matrix components shape the discs' subsequentbiomechanical functions. In another embodiment, the present inventionprovides that by the 2^(nd) week of incubation, the disc tissuedemonstrates resistance to pressure force. In another embodiment, thepresent invention provides that by the 3^(rd) week of incubation, thedisc tissue demonstrates resistance to pressure force. In anotherembodiment, the present invention provides that resistance to pressureforce indicates that the disc is matured. In another embodiment, thepresent invention provides that resistance to pressure force indicatesthat the disc acquired tensile properties. In another embodiment, thepresent invention provides that by the 8^(th) week, the disc tissuedemonstrates maximal thickness and resistance to compressive forces. Inanother embodiment, the present invention provides that by the 9^(th)week, the disc tissue demonstrates maximal thickness and resistance tocompressive forces. In another embodiment, the present inventionprovides that by the 10^(th) week, the disc tissue demonstrates maximalthickness and resistance to compressive forces.

In another embodiment, the present invention provides a method for totaldisc replacement. In another embodiment, the present invention providesa method for partial disc replacement. In another embodiment, the methodfor partial disc replacement comprises replacement of the nucleuspulposus.

In another embodiment, the present invention provides that the ruptureddisc is removed in a minimally invasive manner through a 10-25 mmparaspinal incision. In another embodiment, the present inventionprovides that the ruptured disc is removed in a minimally invasivemanner through a 10-20 mm paraspinal incision. In another embodiment,the present invention provides that the ruptured disc is removed in aminimally invasive manner through a 15-18 mm paraspinal incision. Inanother embodiment, the present invention provides that the ruptureddisc is removed in a minimally invasive manner through a 16-20 mmparaspinal incision.

In another embodiment, the present invention provides that pre-preparedscaffold is inserted into the disc space. In another embodiment, thepresent invention provides that pre-prepared scaffold comprisingcollagen is inserted into the disc space. In another embodiment, thepresent invention provides that pre-prepared scaffold is inserted intothe disc space and expanded to fill the space.

In another embodiment the recipient receives the disc replacement deviceof the present invention. In another embodiment the recipient receivesnucleus pulposus cells of the present invention. In another embodimentthe recipient receives local anesthesia. In another embodiment therecipient receives general anesthesia. In another embodiment the preciseanesthesia protocol will be determined by one of skill in the art.

In another embodiment a damaged disc is removed from the recipient bymethods known to one of skill in the art. In another embodiment, thedisc replacement device of the present invention replaces the damageddisc. In another embodiment, a pre-treated disc scaffold of the presentinvention replaces the damaged disc. In another embodiment, apre-treated disc scaffold of the present invention comprising collagenreplaces the damaged disc. In another embodiment, a pre-treated discscaffold of the present invention comprising various collagens of theinvention replaces the damaged disc. In another embodiment, apre-treated disc scaffold of the present invention comprising varios ECMcomponents replaces the damaged disc.

In another embodiment, nucleus pulposus cells are administered to a discscaffold of the present invention after the disc scaffold is surgicallyplaced in the recipient. In another embodiment, the term “nucleuspulposus cells” comprise disc stem cells, disc progenitor cells,discospheres, or a combination thereof. In another embodiment, nucleuspulposus cells are administered via a blunt tipped needle. In anotherembodiment, nucleus pulposus cells are administered via a cannula. Inanother embodiment, nucleus pulposus cells are forced through theannulus. In another embodiment, nucleus pulposus cells are administeredvia a needle inserted into the anterior or lateral portion of the disc.In another embodiment, one skilled in the art will realize the needlecould be directed into the lateral portion of the disc percutaneouslywith fluoroscopic guidance and into the anterior portion of the disclaparoscopically.

In another embodiment, nucleus pulposus cells of the present inventionare added to the patient's nucleus pulposus. In another embodiment, thepatient's disc is removed with standard techniques. In anotherembodiment, the patient's disc nucleus could be removed with standardenzymatic techniques. In another embodiment, the patient's disc nucleuscould be removed with chymopapain. In another embodiment, the patient'sdisc nucleus could be removed with the aid of a laser. In anotherembodiment, the patient's disc nucleus could be removed with the aid ofa suction device. In another embodiment, the patient's disc nucleuscould be removed with the aid of a shaver. In another embodiment, thepatient's disc nucleus could be removed with the aid of a any otheruseful surgical instrument. In another embodiment, if the nucleus isremoved the hole in the annulus must be small and closed at the end ofthe procedure.

In another embodiment, additional therapeutic substances are added tothe transplanted nucleus. In another embodiment, additional therapeuticsubstances are added to the transplanted disc scaffold. In anotherembodiment, additional therapeutic substances are added to thetransplanted disc replacement device of the present invention.

In another embodiment, additional resorbable culture medium is added tothe transplanted nucleus. In another embodiment, additional tissuegrowth or factors are added to the transplanted nucleus. In anotherembodiment, additional tissue differentiation factors are added to thetransplanted nucleus. In another embodiment, additional recombinantgenerated morphogenetic proteins are added to the transplanted nucleus.In another embodiment, additional PDGF is added to the transplantednucleus. In another embodiment, additional TGF-β is added to thetransplanted nucleus. In another embodiment, additional EGF/TGF-α areadded to the transplanted nucleus. In another embodiment, additionalIGF-I is added to the transplanted nucleus. In another embodiment,additional FGF is added to the transplanted nucleus. In anotherembodiment, additional hydrogels are added to the transplanted nucleus.In another embodiment, additional non-resorbable synthetic or naturalpolymers are added to the transplanted nucleus. In another embodiment,additional collagen is added to the transplanted nucleus. In anotherembodiment, additional fibrin is added to the transplanted nucleus. Inanother embodiment, additional polyglycolic acid is added to thetransplanted nucleus. In another embodiment, additionalpolytetrafluoroethylene is added to the transplanted nucleus. In anotherembodiment, additional antibiotics are added to the transplantednucleus. In another embodiment, additional anti-inflammatory medicationsare added to the transplanted nucleus. In another embodiment, additionalimmunosuppressive medications are added to the transplanted nucleus.

In another embodiment, additional resorbable culture medium is added tothe transplanted disc scaffold. In another embodiment, additional tissuegrowth or factors are added to the transplanted disc scaffold. Inanother embodiment, additional tissue differentiation factors are addedto the transplanted disc scaffold. In another embodiment, additionalrecombinant generated morphogenetic proteins are added to thetransplanted disc scaffold. In another embodiment, additional PDGF isadded to the transplanted disc scaffold. In another embodiment,additional TGF-β is added to the transplanted disc scaffold. In anotherembodiment, additional EGF/TGF-α are added to the transplanted discscaffold. In another embodiment, additional IGF-I is added to thetransplanted disc scaffold. In another embodiment, additional FGF isadded to the transplanted disc scaffold. In another embodiment,additional hydrogels are added to the transplanted disc scaffold. Inanother embodiment, additional non-resorbable synthetic or naturalpolymers are added to the transplanted disc scaffold. In anotherembodiment, additional collagen is added to the transplanted discscaffold. In another embodiment, additional fibrin is added to thetransplanted disc scaffold. In another embodiment, additionalpolyglycolic acid is added to the transplanted disc scaffold. In anotherembodiment, additional polytetrafluoroethylene is added to thetransplanted disc scaffold. In another embodiment, additionalantibiotics are added to the transplanted disc scaffold. In anotherembodiment, additional anti-inflammatory medications are added to thetransplanted disc scaffold. In another embodiment, additionalimmunosuppressive medications are added to the transplanted discscaffold.

In another embodiment, additional resorbable culture medium is added tothe transplanted disc replacement device. In another embodiment,additional tissue growth or factors are added to the transplanted discreplacement device. In another embodiment, additional tissuedifferentiation factors are added to the transplanted disc replacementdevice. In another embodiment, additional recombinant generatedmorphogenetic proteins are added to the transplanted disc replacementdevice. In another embodiment, additional PDGF is added to thetransplanted disc replacement device. In another embodiment, additionalTGF-β is added to the transplanted disc replacement device. In anotherembodiment, additional EGF/TGF-α are added to the transplanted discreplacement device. In another embodiment, additional IGF-I is added tothe transplanted disc replacement device. In another embodiment,additional FGF is added to the transplanted disc replacement device. Inanother embodiment, additional hydrogels are added to the transplanteddisc replacement device. In another embodiment, additionalnon-resorbable synthetic or natural polymers are added to thetransplanted disc replacement device. In another embodiment, additionalcollagen is added to the transplanted disc replacement device. Inanother embodiment, additional fibrin is added to the transplanted discreplacement device. In another embodiment, additional polyglycolic acidis added to the transplanted disc replacement device. In anotherembodiment, additional polytetrafluoroethylene is added to thetransplanted disc replacement device. In another embodiment, additionalantibiotics are added to the transplanted disc replacement device. Inanother embodiment, additional anti-inflammatory medications are addedto the transplanted disc replacement device. In another embodiment,additional immunosuppressive medications are added to the transplanteddisc replacement device.

In another embodiment, additional resorbable culture medium is added tothe transplanted discospheres, disc stem cells, disc progenitor cells,or a mixture thereof of the present invention. In another embodiment,additional tissue growth or factors are added to the transplanteddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof of the present invention. In another embodiment, additionaltissue differentiation factors are added to the transplanteddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof of the present invention. In another embodiment, additionalrecombinant generated morphogenetic proteins are added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional PDGF is added to the transplanted discospheres, disc stemcells, disc progenitor cells, or a mixture thereof of the presentinvention. In another embodiment, additional TGF-β is added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional EGF/TGF-α are added to the transplanted discospheres, discstem cells, disc progenitor cells, or a mixture thereof of the presentinvention. In another embodiment, additional IGF-I is added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional FGF is added to the transplanted discospheres, disc stemcells, disc progenitor cells, or a mixture thereof of the presentinvention. In another embodiment, additional hydrogels are added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional non-resorbable synthetic or natural polymers are added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional collagen is added to the transplanted discospheres, disc stemcells, disc progenitor cells, or a mixture thereof of the presentinvention. In another embodiment, additional fibrin is added to thetransplanted discospheres, disc stem cells, disc progenitor cells, or amixture thereof of the present invention. In another embodiment,additional polyglycolic acid is added to the transplanted discospheres,disc stem cells, disc progenitor cells, or a mixture thereof of thepresent invention. In another embodiment, additionalpolytetrafluoroethylene is added to the transplanted discospheres, discstem cells, disc progenitor cells, or a mixture thereof of the presentinvention. In another embodiment, additional antibiotics are added tothe transplanted discospheres, disc stem cells, disc progenitor cells,or a mixture thereof of the present invention. In another embodiment,additional anti-inflammatory medications are added to the transplanteddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof of the present invention. In another embodiment, additionalimmunosuppressive medications are added to the transplanteddiscospheres, disc stem cells, disc progenitor cells, or a mixturethereof of the present invention.

In another embodiment, a matrix formulated disc stem cell preparationloaded with key nutrients is injected into the disc space and will growinto a disc tissue structure over time restoring the damaged disc(Example 4). In another embodiment, a matrix formulated disc stem cellpreparation further comprises resorbable culture medium. In anotherembodiment, a matrix formulated disc stem cell preparation furthercomprises tissue growth or factors. In another embodiment, a matrixformulated disc stem cell preparation further comprises tissuedifferentiation factors. In another embodiment, a matrix formulated discstem cell preparation further comprises recombinant generatedmorphogenetic proteins. In another embodiment, a matrix formulated discstem cell preparation further comprises PDGF. In another embodiment, amatrix formulated disc stem cell preparation further comprises TGF-β. Inanother embodiment, a matrix formulated disc stem cell preparationfurther comprises EGF/TGF-α. In another embodiment, a matrix formulateddisc stem cell preparation further comprises IGF-I. In anotherembodiment, a matrix formulated disc stem cell preparation furthercomprises FGF. In another embodiment, a matrix formulated disc stem cellpreparation further comprises hydrogels. In another embodiment, a matrixformulated disc stem cell preparation further comprises non-resorbablesynthetic or natural polymers. In another embodiment, a matrixformulated disc stem cell preparation further comprises collagen. Inanother embodiment, a matrix formulated disc stem cell preparationfurther comprises fibrin. In another embodiment, a matrix formulateddisc stem cell preparation further comprises polyglycolic acid. Inanother embodiment, a matrix formulated disc stem cell preparationfurther comprises polytetrafluoroethylene. In another embodiment, amatrix formulated disc stem cell preparation further comprisesanti-inflammatory medications. In another embodiment, a matrixformulated disc stem cell preparation further comprises antibiotics. Inanother embodiment, a matrix formulated disc stem cell preparationfurther comprises immunosuppressive medications.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc, comprising the step ofadministering to a subject an artificial disc comprising nucleuspulposus cells. In another embodiment, the subject is a human subject.In another embodiment, the subject is a farm animal. In anotherembodiment, the subject is a pet animal.

In another embodiment, the present invention provides that administeringto a subject an artificial disc comprises transplanting to a subject anartificial disc. In another embodiment, the present invention providesthat the replacement device comprises processed biological tissues froma single donor. In another embodiment, the present invention providesthat the replacement device comprises processed biological tissues froma single donor which is the patient in need of an artificial disc. Inanother embodiment, the present invention provides that the replacementdevice comprises processed biological tissues in combination with menmade materials. In another embodiment, the present invention providesthat the replacement device comprises processed biological tissues incombination with plastic based materials. In another embodiment, thepresent invention provides that the replacement device comprisesprocessed biological tissues in combination with ceramics. In anotherembodiment, the present invention provides that the replacement devicecomprises processed biological tissues in combination with metals.

In another embodiment, the present invention provides a method oftreating a subject having a herniated disc. In another embodiment, thepresent invention provides a method of treating a subject having adegenerative disc disease (DDD). In another embodiment, the presentinvention provides a method of treating a subject having a DDD at onelevel in the lumbar spine (from L3-S1). In another embodiment, thepresent invention provides a method of treating a subject having no morethan Grade 1 spondylolisthesis. In another embodiment, the presentinvention provides a method of treating a subject having more than Grade1 spondylolisthesis. In another embodiment, the present inventionprovides a method of treating a subject having no more than Grade 1spondylolisthesis that have had no relief from pain after at least sixmonths of non-surgical treatment.

In another embodiment, the present invention provides that administeringto a subject an artificial disc restores disc height. In anotherembodiment, the present invention provides that administering to asubject an artificial disc may reduce pain. In another embodiment, thepresent invention provides that administering to a subject an artificialdisc restores movement at the level where it is implanted. In anotherembodiment, the present invention provides posterolateral annulotomyafter discectomy.

EXPERIMENTAL DETAILS SECTION Materials and Methods

Methylcellulose-Based Medium for Expanding Disc Stem/Progenitor Cellsinto Discospheres Comprising

The methylcellulose-based (medium for expanding discospheres comprisingdisc stem/progenitor cells contained a base DMEM/F12 medium supplementedwith 2% Methylcellulose, 10 μg/ml insulin, 40 nM progesterone, 200 μMputrescine, 100 μg/ml transferrin, 60 nM sodium selenite, 10 ng/mlrecombinant FGF2, and 10 ng/ml recombinant EGF.

Methylcellulose-Based Medium for Expanding Discospheres Comprising DiscStem/Progenitor Cells

The Methylcellulose-based medium for expanding discospheres comprisingdisc stem/progenitor cells contained a base DMEM/F12 medium supplementedwith 0.8% Methylcellulose, 5 μg/ml insulin, 20 nM progesterone, 100 μMputrescine, 50 μg/ml transferrin, and 30 nM sodium selenite. 10 ng/mlFGF2 and 10 ng/ml EGFb were added every 3^(rd) day.

Histochemistry

Hematoxilin-Eosin Staining

Hematoxilin-Eosin staining on disc biopsies obtained from the discsproduced by the procedures disclosed in Example 3 were preformed asfollows: Fonmalin fixed paraffin embedded tissue sections (5 μm) weresequentially deparaffinized and rehydrated. Then slides were stainedwith Harris' haematoxylin for 10 minutes, washed and blue in running tapwater for 1 minute, differentiated in acid alcohol (1% hydrochloric acidin 70% alcohol) for 10 seconds, washed and blue in running tap water for5 minutes, stained with eosin for 4 minutes, and finally washed in tapwater, dehydrated through graded alcohol and cleared in xylene.

von Kossa Staining

von Kossa Staining on disc biopsies obtained from the discs produced bythe procedures disclosed in Example 3 were preformed as follows:Formalin fixed paraffin embedded tissue sections (5 μm) weresequentially deparaffinized and rehydrated. Sections were incubated with1% silver nitrate solution in a clear glass coplin jar placed underultraviolet light for 20 minutes. Then sections were rinsed in severalchanges of distilled water followed by the removal of un-reacted silverwith 5% sodium thiosulfate for 5 minutes. Then sections were rinsed inseveral changes of distilled water and counterstained with nuclear fastred for 5 minutes. Finally, sections were rinsed in several changes ofdistilled water, dehydrated through graded alcohol and cleared inxylene.

Immunohistochemical Identification of Collagen Type I, Collagen Type II,or Ki67 in Tissue

Immunohistochemical staining for collagen type I, collagen type H, orKi67 on disc biopsies from Example 3 were preformed as follows: Formalinfixed paraffin embedded tissue sections (5 μm) were sequentiallydeparaffinized, rehydrated, and blocked for endogenous peroxidaseactivity following a 95° C. degree, 25 minutes antigen retrieval inTrilogy unmasking solution (Cell Marque, Hot Springs Ark.). Slides werebiotin blocked, serum blocked and immunostained using a goat ABC EliteKit (Vector Labs, Burlingame, Calif.) Antibodies to collagen type I(cat. #: 63170, MP Biomedicals, Solon, Ohio), collagen type II (cat. #:MAB 1330, Chemicon, Billerica, Mass.), or Ki67 (cat. #: MAB4062,Chemicon, Billerica, Mass.) were applied at 1:100 dilution for one hourat room temperature. Positive staining was detected with DAB(3,3′-Diaminobenzidene). Immuno-reactivity was visualized with a Bio-Radconfocal microscope and images collected on a computer for lateranalysis.

Safranin O Staining for Cartilage

This method was used for the detection of cartilage on formalin-fixed,paraffin-embedded tissue sections. The cartilage was stained orange tored, and the nuclei will were stained black. The background was stainedgreen. Weigert's Iron Hematoxylin Solution was prepared from two stocksolutions. Stock Solution A: 1 g Hematoxylin, 100 ml 95% alcohol. StockSolution B: 4 ml 29% Ferric chloride in water, 95 ml distilled water, 1ml Hydrochloric acid. Equal parts of stock solution were mixed resultingin Weigert's Iron Hematoxylin Solution.

0.1% Safranin O Solution was prepared by mixing 0.1 g Safranin O, C.I.50240 and 100 ml distilled water. Then slides were deparaffinized andhydrated to distilled water followed by staining the slides withWeigert's iron hematoxylin working solution for 10 minutes. Followed bywashing the slides in running tap water for 10 minutes and staining withfast green (FCF) solution for 5 minutes, rinsing quickly with 1% aceticacid solution for 10 seconds, and staining in 0.1% safranin O solutionfor 5 minutes. Slides were then dehydrated and cleared with 95% ethylalcohol, absolute ethyl alcohol, and xylene, using 2 changes each, 2minutes each. Finally slides were mounted using resinous medium.

Example 1 A Method of Growing Discospheres

A biopsy specimen of human nucleus pulposus was minced into piecesapproximately 2-3 millimeters in size and transferred to a 50 ml falcontube containing 30 ml of Phosphate buffered saline (PBS) supplementedwith standard antibiotics and antimycotics (standardpenicillin/streptomycin solution (GIBCO BRL) in concentration 1:100).

PBS was aspirated and 30 ml of Dulbecco's Modified Eagle Media with F12(DMEM/F12) medium containing 300 U/ml of Collagenase II solution wasadded to the 50 ml tube.

The tube was placed in a horizontal position in a shaker incubator at37° C. at 100 RPM for 2-3 hours until fragments were completelydissociated.

The cell suspension was filtered through a nylon mesh into a 50 mlfalcon tube and triturated with a fire-polished pasteur pipette to forma single-cell suspension. A cell count was performed at this point todetermine the cell concentration.

The cell suspension was then centrifuged at room temperature for 4minutes (min.) at 400 g, followed by the removal of the supernatant byaspiration.

Cells were resuspended in DMEM/F12 medium supplemented with insulin (10ug/ml), progesterone (40 nM), putrescine (200 uM), transferrin (100ug/ml), sodium selenite (60 nM) to a final density of 120,000 cells/ml.

A volume of a 2% solution of methylcellulose in DMEM/F12 medium equal tofinal volume obtained previously was added to the cell suspension andmixed by vortexing.

Growth factors EGF and FGF2 were added to final concentration 10 ng/mland mixed again.

Finally, the cell/media suspension was added to 6-well plates atapproximately 2 ml/well comprising about 120,000 cells per well, andincubated at 37° C. in 5% CO2. Each well was pre-coated with ananti-adhesive substance (e.g. poly 2-hydroxyethyl methacrylate (#P-3932Sigma) anti-adhesive coating) according to manufacturer'srecommendations.

Growth factors were added every 3^(rd) day.

After approximately 2 weeks, discospheres had formed in the culture.

Example 2 A Method of Expanding Discospheres Cell Culture

Discospheres obtained by the method disclosed in Example 1 weredissociated by incubation at 37° C. in DMEM/F12 medium supplemented bycollagenase II (300 U/ml).

Dissociated cells were expanded in 6-well plates according by passagingthe cells using the same plating and culture techniques as described inExample 1.

Example 3 A Method of Obtaining a Spinal Disc Collagen Scaffold(Annulus)

A postmortem (rabbit cadaver) intervertebral disc was removed bydissection with the vertebral endplates left intact. The intervertebraldisc sample was soaked in 4 M guanidine thyocyonate for 24 hours at roomtemperature to remove intradisc biomaterial. After 24 hours, theintradisc biomaterial was liquidfied.

The liquid was aspirated, and the remaining disc scaffold was washed 3times with room temperature PBS.

At this stage the disc scaffold can be stored in PBS at 4° C. up to oneyear.

Example 4 A Method of Obtaining an Artificial Disc

The disc scaffold obtained according to the method disclosed in Example3 was placed in tissue culture vessel and washed 3 times with DMEM/F12medium with 10% FCS and incubated at 37° C. in 5% CO2 for 8 hours.

Discospheres were pooled from culture and collected in DMEM/F12. Thendiscospheres were washed free of methylcellulose with DMEM/F12, andsuspended in 200 μl DMEM/F12 medium.

The suspended discosphere were injected into the center of the scaffoldincubated at 37° C. in 5% CO2 for 8 hours.

The disc tissue culture vessel was then filled with DMEM/F12 medium andincubated at 37° C. in 5% CO2.

The media was changed every 3^(rd) day.

Results

Nucleus pulposus cells were harvested from a donor patient and preparedas a single cell suspension as described in Example 1. Afterapproximately 2 weeks, discospheres were collected and prepared forinjection into the pre-processed rabbit annulus fibrosis. This discscaffold containing the disc stem cell preparation was then placed in atissue culture vessel for 3 months. The media was changed every thirdday. Each day, a downward pressure was applied to each disc tissue toinduce biomechanical regulated differentiation programs.

Biomechanical Properties

Disc cells laid down extracellular matrix components of the disc, whichin turn, shaped the discs' subsequent physiological and biomechanicalfunctions. By the 3^(rd) week, the disc tissue began to demonstrateresistance to pressure force, indicating its maturation and acquisitionof tensile properties. By the 10th week, the disc tissue demonstratedmaximal thickness and resistance to compressive forces.

Comparative Histology

After 3 months of culture, the disc tissues were removed from cultureand sectioned with a cryostat. Basic histological analyses werecompleted using selected tissue stains and immunohistochemistry.

As shown in FIG. 1 (Panel 1), Hematoxylin-Eosin staining revealed thatthe gross structure and cellular morphology of the human disc tissuegrown from disc stem cells was comparative to that derived from healthyrabbit disc tissue. Additionally, safranin staining (FIG. 1, Panel 2)demonstrated that a rich cartilage matrix of sulfated proteoglycans wassecreted into the extracellular matrix by the disc stem cells and wascomparable to healthy rabbit disc tissue at the time of analysis. VonKossa staining (FIG. 1, Panel 3) demonstrated the absence of anyosteogenic differentiation of in vitro disc stem cells in this culturesystem. Finally, immunohistochemical staining with collagen type II(FIG. 2) and type I (FIG. 3) demonstrated high and low expressionrespectively indicating maturation of the disc tissue and again wasfound to be comparable to healthy controls.

Demonstration of Lack of Proliferation in the Tissue

As a further indicator that the disc tissue was mature and thus did notcontain any immature and/or proliferating cells, Ki67 (marker ofproliferation) immunostaining was performed on the tissues. As shown inFIG. 4, no proliferating cells were noted in the control tissues or thedisc tissue grown from human disc stem cells.

1. A composition comprising a disc stem cell population wherein saiddisc stem cell population is from a nucleus pulposus of a subject,wherein said composition further comprises a serum-free culture mediacomprising fibroblast growth factor 2 (FGF2), epidermal growth factor(EGF), stem cell factor (SCF), interleukin-6 (IL-6), interleukin-2(IL-2), transforming growth factor-β (TGF-β) leukemia inhibitory factor(LIF), or a combination thereof.
 2. The composition of claim 1, whereinsaid disc stem cells are human disc stem cells.
 3. The composition ofclaim 1, wherein said composition further comprises disc progenitorcells.
 4. The composition of claim 1, wherein said disc stem cellpopulation forms discospheres when grown in suspension in tissue culturevessels coated with an anti-adhesive substrate.
 5. The composition ofclaim 1, wherein said media additionally comprises insulin,progesterone, putrescine, transferrin, sodium selenite, or a combinationthereof.
 6. A composition comprising a discosphere, wherein saidcomposition further comprises a serum free media.
 7. The composition ofclaim 6, wherein said media further comprises FGF2, EGF, SCF, IL-6,IL-2, TGF-β, LIF, or a combination thereof.
 8. The composition of claim6, wherein said media additionally comprises insulin, progesterone,putrescine, transferrin, sodium selenite, or a combination thereof.